This application is concerned with a novel selectable marker, especially with the use of cyanamide hydratase as a selection marker in transformation experiments, more specifically in plant transformation experiments.
Cyanamide (H2Nxe2x80x94Cxe2x89xa1N) is a nitrile derivate which, like other nitrile derivates, is used in agriculture for stimulation of growth and for plant protection. Cyanamide in aqueous solution or in the form of its calcium salt is used as a fertilizer by providing ammonia to the soil by its metabolic conversion. It has, however, the additional advantage of acting as a herbicide. To use it as fertilizer it has to be applied before sowing.
Chemically, cyanamide belongs to the class of nitrites. In spite of the relatively rare occurrence in nature of compounds containing the nitrile group, enzymes that hydrate this group have been found in bacteria and plants (e.g. Nagasawa T., et al. (1988) Biochem. Biophys. Res. Commun. 155 1008-1016; Endo T. and Watanabe I. (1989) FEBS Lett. 243 61-64). Also in the fungus Myrothecium verrucaria a nitrile hydrating enzyme was found (Stransky H. and Amberger A. (1973) Z. Pflanzenphysiol. 70 74-87), which hydrates the nitrile group of cyanamide with formation of urea:
H2Nxe2x80x94Cxe2x89xa1N+HOH= greater than H2Nxe2x80x94COxe2x80x94NH2
Maier-Greiner et al. have isolated the enzyme and cloned the gene coding for it (Proc. Natl. Acad. Sci. USA 88, 4260-4264, 1991). They have demonstrated that this enzyme shows an extremely narrow substrate specificity, where compounds chemically related to cyanamide are not recognized as substrates.
Selectable markers have to confer a dominant phenotype on transformed cells which is able of being used as a selection criteria. These fall into two classes: one class of genes which confers either cell viability or lethality in the presence of a selective agent and a class of genes which has negligible effects on cell survival but which confers transformed cells with some distinguishing physical characteristic.
In plant transformation the fraction of cells incorporating the novel DNA is generally low, so most stable transformation schemes use markers which ensure the survival of transformed cells in the presence of a selective agent.
A number of selection markers of this first group has been known and used for plant transformation experiments for several years. Included are the enzyme neomycin phosphotransferase (npt) which confers resistance to a group of antibiotics including kanamycin, paromomycin, geneticin and neomycin, mutant forms of the enzyme acetolactate synthase (als) which confer resistance to imidazolinones, sulfonylureas, triazolopyrimidines and pyrimidyloxybenzoates and the enzyme hygromycin 3xe2x80x2-O-phosphotransferase (hpt) which confers resistance to hygromycin. Also available are chloramphenicol transferase (cat) which detoxifies chloramphenicol and dihydrofolate reductase (dhfr) which neutralizes the toxic effects of methotrexate. Another possibility is to use the bar gene for resistance to the herbicide bialaphos (WO 97/05829).
Although there already are a number of selectable markers available, there is still need for another marker. This is due to several reasons:
when transgenic plants are being transformed for a second time with a new construct it is necessary to select for the newly formed transformants with the help of a second selectable marker.
the above mentioned selection markers are not applicable on in all plant species.
some of the compounds which have to be added to enable selection are antibiotics. Spreading of genes which give resistance to antibiotics or herbicides should be minimized as much as possible to avoid the risk of conferring resistance to pathogens.
some of the compounds which have to be added to enable selection are relatively expensive. There is a need for cheaper selection agents.
The invention now provides the use of a gene coding for cyanamide hydratase (CAH) as a new selection marker. Preferably this can be used for the transformation of plants. The gene comprises the nucleotide sequence of SEQIDNO: 1 or muteins thereof having cyanamide hydratase function.
The invention further comprises a method for the selection of transformed plants which comprises constructing a vector carrying a coding sequence for CAH and a gene of interest, transforming the vector to plants or plant parts or plant cells or callus and growing the resulting transformants in a medium which comprises cyanamide.
The invention is also directed to the use of cyanamide for the selection of plants transformed with a gene coding for CAH.
Further part of the invention are expression cassettes comprising a nucleotide sequence coding for cyanamidehydratase and a gene of interest. Also part of the invention are vectors with this expression cassette and hosts, including Agrobacterium, harboring such a vector. Further, plants transformed with such a vector and/or such an Agrobacterium form part of the invention.